Department of Ophthalmology, University of Florida, Gainesville, FLDepartment of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL

Sanford L Boye

Department of Ophthalmology, University of Florida, Gainesville, FL

Frank M Dyka

Department of Ophthalmology, University of Florida, Gainesville, FL

Kathryn MH Fransen

Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, KY

Charles N de Leeuw

Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Colombia, Vancouver, BC, CanadaDepartment of Medical Genetics, University of British Colombia, Vancouver, BC, Canada

Elizabeth M. Simpson

Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Colombia, Vancouver, BC, CanadaDepartment of Medical Genetics, University of British Colombia, Vancouver, BC, Canada

Ronald G Gregg

Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY

Maureen A McCall

Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, KY

Purpose:
Congenital Stationary Night Blindness (CSNB) is an inherited retinal disorder characterized by the inability to see in low light conditions. Patients also have difficulties seeing in daylight conditions due to a high frequency of myopia, nystagmus, and strabismus. In the complete form of this disease (CSNB1), signaling from photoreceptors to ON bipolar cells (ON BCs) is disrupted due to mutations in genes encoding post synaptic proteins involved in the metabotropic glutamate receptor 6 (mGluR6) G-protein coupled cascade. Despite this signaling dysfunction, the retinas of CSNB1 patients and mouse models do not degenerate. One such gene, NYX, encodes Nyctalopin and its mutated form is associated with X-linked CSNB1. Without functional NYX, TRPM1 cation channel is mislocalized and cannot be gated. Using a novel serotype/promoter combination, we have established an intravitreal method of delivery for AAV-mediated transduction of ON BCs. We evaluated the potential of this tool to restore mGluR6-mediated signaling in ON BCs in a model of XCSNB1, the nyxnob mouse.

Methods:
A construct containing an ON BC specific promoter, Ple155, driving YFP_Nyx was packaged into an AAV2(quadY-F+T-V). P2 or P30 nyxnob mice were intravitreally injected with 6.5E12 VG. Electoretinograms (ERG) and whole cell patch clamp responses were recorded from injected and control eyes ~4 weeks post injection. Retinas were examined using immunohistochemistry and confocal microscopy.

Results:
Following P30 treatment, NYX expression was limited and no measurable improvement in the ERG b-wave was detected. Eyes injected at P2, showed “robust” NYX expression exclusively in ON BCs, restoration of scotopic and photopic ERG b-waves and gating of the TRPM1 channel both directly and via the mGluR6 cascade (capsaicin and CPPG responses).

Conclusions:
This study is the first demonstration of therapy following gene replacement in a model of ON BC-mediated disease and provides proof of concept for an AAV-based strategy to treat CSNB1.